Increased trophoblastic apoptosis mediated by neutrophil gelatinase-associated lipocalin (NGAL) activation in the nitrofen model of congenital diaphragmatic hernia.

BACKGROUND: Retinoids play a key role in fetal lung development. It has been suggested that the maternal-fetal retinol transport is disrupted by trophoblastic apoptosis. The mechanism underlying nitrofen-induced apoptosis in placenta is not fully understood. Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in the fetal part of the maternal-fetal interface. NGAL is part of the immune barrier and serves primarily as a transport protein transferring biologically hazardous molecules in a safe and controlled way. It has been shown that over-activation of NGAL induces apoptosis. We hypothesized that increased placental NGAL expression induces trophoblastic apoptosis in the nitrofen model of CDH. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Placenta harvested on D21 and divided into two groups: control and nitrofen with CDH. Immunohistochemistry was performed to evaluate trophoblasts (by cytokeratin expression), NGAL expression, and apoptotic trophoblastic cells (using TUNEL assay). Total RNA was extracted from each placenta and the relative mRNA expression levels of NGAL were analyzed using RT-PCR. RESULTS: Immunohistochemistry showed NGAL immunoreactivity both in control and CDH in the fetal part of the fetal-maternal interface of placenta. Markedly increased NGAL expression was detected in CDH group compared to controls. Relative mRNA expression levels of NGAL gene were significantly increased in the CDH group compared to control in the placenta (5.924 ± 0.93 vs. 1.895 ± 0.54, p < 0.001). Markedly increased numbers of apoptotic trophoblastic cells were seen in the maternal-fetal interface in the CDH group compared to controls. CONCLUSIONS: NGAL activation may lead to increased trophoblastic apoptosis in the maternal-fetal interface in the nitrofen model of CDH. These changes may therefore cause disturbance in maternal-fetal retinol transport affecting fetal lung morphogenesis.

Increased activation of NADPH oxidase 4 in the pulmonary vasculature in experimental diaphragmatic hernia.

AIM: Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). NADPH oxidases (Nox) are the main source of superoxide production in vasculature. Nox4 is highly expressed in the smooth muscle and endothelial cells of the vascular wall and increased activity has been reported in the pulmonary vasculature of both experimental and human pulmonary hypertension. Peroxisome proliferator-activated receptor (PPARγ) is a key regulator of Nox4 expression. Targeted depletion of PPARγ results in pulmonary hypertension phenotype whereas activation of PPARγ attenuates pulmonary hypertension and reduces Nox4 production. The nitrofen-induced CDH model is an established model to study the pathogenesis of pulmonary hypertension in CDH. It has been previously reported that PPARγ-signaling is disrupted during late gestation and H(2)O(2) production is increased in nitrofen-induced CDH. We designed this study to investigate the hypothesis that Nox4 expression and activation is increased and vascular PPARγ is decreased in nitrofen-induced CDH. METHODS: Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into control and CDH. RT-PCR, western blotting and confocal-immunofluorescence-double-staining were performed to determine pulmonary expression levels of PPARγ, Nox4 and Nox4-activation (p22(phox)). RESULTS: There was a marked increase in medial and adventitial thickness in pulmonary arteries of all sizes in CDH compared to controls. Pulmonary Nox4 levels were significantly increased whereas PPARγ levels were decreased in nitrofen-induced CDH compared to controls. Western blotting revealed increased pulmonary protein expression of the Nox4-activating subunit p22(phox) and decreased protein expression of PPARγ in CDH compared to controls. Confocal-microscopy confirmed markedly increased pulmonary expression of the Nox4 activating subunit p22(phox) accompanied by decreased perivascular PPARγ expression in lungs of nitrofen-exposed fetuses compared to controls. CONCLUSION: To our knowledge, the present study is the first to report increased Nox4 production in the pulmonary vasculature of nitrofen-induced CDH. Down-regulation of the PPARγ-signaling pathway may lead to increased superoxide production, resulting in pulmonary vascular dysfunction and contributing to pulmonary hypertension in the nitrofen-induced CDH model. PPARγ-activation inhibiting Nox4 production may therefore represent a potential therapeutic approach for the treatment of pulmonary hypertension in CDH.

Nitrofen interferes with trophoblastic expression of retinol-binding protein and transthyretin during lung morphogenesis in the nitrofen-induced congenital diaphragmatic hernia model.

BACKGROUND: Retinoids play a key role in lung development. Retinoid signaling pathway has been shown to be disrupted in the nitrofen model of congenital diaphragmatic hernia (CDH) but the exact mechanism is not clearly understood. Retinol-binding protein (RBP) and transthyretin (TTR) are transport proteins for delivery of retinol to the tissues via circulation. Previous studies have shown that pulmonary retinol levels are decreased during lung morphogenesis in the nitrofen CDH model. In human newborns with CDH, both retinol and RBP levels are decreased. It has been reported that maternal RBP does not cross the placenta and the fetus produces its own RBP by trophoblast. RBP and TTR synthesized in the fetus are essential for retinol transport to the developing organs including lung morphogenesis. We hypothesized that nitrofen interferes with the trophoblastic expression of RBP and TTR during lung morphogenesis and designed this study to examine the trophoblastic expression of RBP and TTR, and the total level of RBP and TTR in the lung in the nitrofen model of CDH. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs and placenta harvested on D21 and divided into two groups: control (n = 8) and nitrofen with CDH (n = 8). Total lung RBP and TTR levels using protein extraction were compared with enzyme linked immunoassay (ELISA). Immunohistochemistry was performed to evaluate trophoblastic RBP and TTR expression. RESULTS: Total protein levels of lung RBP and TTR were significantly lower in CDH (0.26 ± 0.003 and 6.4 ± 0.5 µg/mL) compared with controls (0.4 ± 0.001 and 9.9 ± 1.6 µg/mL, p < 0.05). In the control group, immunohistochemical staining showed strong immunoreactivity of RBP and TTR in the trophoblast compared to CDH group. CONCLUSIONS: Decreased trophoblast expression of retinol transport proteins suggest that nitrofen may interfere with the fetal retinol transport resulting in reduced pulmonary RBP and TTR levels and causing pulmonary hypoplasia in CDH.

TLR4 influences the humoral and cellular immune response during polymicrobial sepsis.

As part of the innate immune system, Toll-like receptors (TLRs) react rapidly on a pathogen challenge without prior exposure. Although it is well known that TLR4 is associated with the receptor for lipopolysaccharide (LPS), its role during sepsis has not yet been clearly defined. To study this,polymicrobial sepsis was induced in male C3H/HeN (TLR4 wild type) and C3H/HeJ (TLR4 mutant) mice by caecal ligation and puncture (CLP).A total of 48 h following the surgical procedure, the mice were sacrificed and plasma was collected.Kupffer cells were isolated and ex vivo cytokine production and plasma levels were determined. Lung neutrophil influx was investigated by myeloperoxidase (MPO) content and immunohistochemistry. T-cell subtypes in blood and spleen were determined by flow cytometry.Mice with intact TLR4 (wild type) had increased Kupffer cell IL-6 production and increased plasma levels as compared with C3H/HeJ mice following sepsis. Furthermore, wild type mice showed increased neutrophil influx in lungs and lower percentages of CD8+ splenocytes. This was accompanied with less activity, increased weight loss and decreased core temperature.We conclude that TLR4 influences the humoral and cellular response during the course of sepsis and lack of TLR4 reduces markers of the systemic inflammatory response as well as distant organ damage.Therefore, TLR4 could act as a future therapeutic target modulating the immune response during sepsis.

Thoracoscopy versus thoracotomy improves midterm musculoskeletal status and cosmesis in infants and children.

BACKGROUND: It has been postulated that video-assisted thoracoscopic surgery (VATS) achieves a better biometric and aesthetic outcome than conventional thoracic surgery (CTS), but data are lacking. We aimed to compare the midterm effects of both approaches in children. METHODS: Sixty-two infants and children, who underwent VATS (34; 55%) or CTS (28; 45%) for benign thoracic conditions, were evaluated at follow-up after a mean of 3.8 years (1 to 7 years). The patients underwent standardized clinical assessment of the skeletal system and function. The intercostal spaces were investigated for rib fusion by ultrasound. Patients (+/- parents) themselves, as well as clinicians, subsequently assessed the scars. RESULTS: Comparing the operated versus nonoperated sides, chest asymmetry was significantly less frequent after VATS versus CTS in the horizontal plane (mean relative difference 0.996 +/- 0.003 vs 0.964 +/- 0.008, p < 0.001) and in nipple location (mean relative difference 0.985 +/- 0.008 vs 0.949 +/- 0.013, p = 0.047). The ranges of motion of the shoulder joints did not differ significantly. However, the incidence of scoliosis was lower in VATS patients (9% vs 54%, p < 0.001) and the intercostal spaces of the operated hemithoraces were narrower after CTS (p < 0.001). The Manchester scar assessment scores were in favor of VATS (mean 7.5 vs 13.1, p < 0.001). The visual analog scale scores recorded by patients-parents and independent observers were also significantly better after VATS. Patient satisfaction was less with CTS as 10% wanted to have the scar revised, compared with none in the VATS group. CONCLUSIONS: The thoracoscopic versus conventional approach to the thoracic cavity in children is associated with significantly less midterm musculoskeletal sequelae and a better cosmetic outcome.